[SRM] Full-length cDNA clones for 16 selected proteins were obtained from the pANT7_cGST clone collection distributed by the Arizona State University Biodesign Institute plasmid repository. Each bacterial stock clone was grown independently overnight in 5 ml of Luria-Bertani broth with 100 μg ml−1 ampicillin (LB-amp). Plasmid DNA was extracted using the manufacturer’s spin mini-prep protocol (QIAGEN). Proteins were then synthesized from plasmid DNA using the Pierce Human in vitro Protein Expression kit (Thermo) according to the manufacturer's protocol with GFP control. We then enriched the GST-fusion proteins using glutathione sepharose 4B beads (GE) with a published method19. Finally, these enriched GST-fusion proteins were reduced, alkylated, and digested for 2 h with trypsin individually. Ninety-one peptides were selected for the 16 proteins based on a preliminary analysis of PECAN during its early development. Each protein digestion was injected separately and analyzed with a TSQ-Vantage triple-quadrupole instrument (Thermo) using a nanoACQUITY UPLC (Waters). A 3-μl aliquot of sample was loaded for a 27.5-min LC setting. Ions were isolated in both Q1 and Q3 using 0.7 FWHM resolution. Peptide fragmentation was performed at 1.5 mTorr in Q2 without peptide specific collision energies. Data was acquired using a scan width of 0.002 mass to charge ratio (m/z) and a dwell time of 10 ms. [DIA] HeLa protein digest (Thermo) spiked-in with stable-isotope labeled peptides (PRTC, Thermo) was analyzed on a Q-Exactive HF mass spectrometer. One μg of HeLa peptides and 40 fmol of PRTC was loaded in each injection and separated with a 90-min linear gradient LC. Four gas-phase fractionation (GPF) injections covering 500-600, 600-700, 700-800, and 800-900 precursor m/z (noted as 4xGPF in the manuscript). 5 m/z-wide isolation windows was used to acquire 4xGPF DIA respectively. For FDR control, data from multiple injections were analyzed together as if they were from one instrument run.